Abstract

Cytoplasmic osmolytes can significantly alter the thermodynamic and kinetic properties of proteins relative to those under dilute solution conditions. Spectroscopic experiments of lysozymes in cosolvents indicate that such changes may arise from the heterogeneous, site-specific hydrophobic interactions between protein surface residues and individual solvent molecules. In pursuit of an accurate and predictive model for explaining biomolecular interactions, we study the averaged structural characteristics of mixed solvents with homologous lysozyme solutes using all-atom molecular dynamics. By observing the time-averaged densities of different aqueous solutions of trifluoroethanol, we deduce trends in the heterogeneous solvent interactions over each protein's surface, and investigate how the homology of protein structure does not necessarily translate to similarities in solvent structure and composition-even when observing identical side chains.

(A) Percent TFE v/v calculated for the local environment of each surface-lying residue. Shown here are the average percentage of TFE for α-helices (green) and unstructured regions of the protein (magenta). α-Helices show a local increase in TFE concentration relative to bulk solvent (gray/black), while unstructured regions show a relatively bulk-like concentration. The error bars are the standard deviation among the three parallel trajectories for each protein at each concentration. (B) HEWL is shown as a visual cue for the general distribution and location of high density hot spots. TFE (red) and water (cyan) hotspots do not overlap in this study. (C) The total volume of hot spots for water and TFE exhibit a crossover near 10% TFE, beyond which the majority of hot spots are due to TFE. The error bars are the standard deviation among the three parallel trajectories for each protein at each concentration.

Local solvent structures near the histidines in simulations of 10% TFE v/v. (A) HEWL (yellow) with histidine 15 (orange sticks) is surrounded by TFE (red) and water (blue) isosurfaces. (B) Isosurfaces for histidine 78 on humLys. Notice that both locations are surrounded by similar ratios of both solvent types. (C) The average number of empty voxels in the local environment around each histidine at various cosolvent concentrations as calculated by integrating G(r) functions. The error bars correspond to the standard deviations of data among the three independent simulations at each concentration of TFE.

For all plots, only data from solvent-exposed amino acids are considered. Panels A–D show average correlation coefficients between amino acids of one protein (HEWL or humLys) in solutions of different concentrations of TFE v/v. All correlations fall between 1 (on the diagonals) and 0.54 (at the corners) in these plots. Plots A and C are correlations of water densities at different concentrations, and plots B and D are correlations of TFE. Plot E is an average correlation of G(r) functions around each amino acid by comparing residues from HEWL to its homologue on humLys. The error bars are the standard deviation of data among the correlations of amino acids. Plot F is the same analysis as seen in plot E, except for only residues on the α-helix that has 100% conservation of residue identity. A stronger correlation is observed here, but due to neighboring effects of nonidentical amino acids, the TFE distributions remained nonhomologous between the proteins.

All three figures above show a reference lysozyme tertiary structure (yellow) and the residues of the α-helix that are conserved between hen egg white and human lysozymes (orange). Since this study ignores buried residues, only the surface-lying residues 107, 108, 109, 112, 113, and 114 are shown as sticks. Panel A illustrates the configuration of side chains, and panels B and C overlay the protein with solvent density averaged from the three replicas at 10% TFE. Even though this helix is completely conserved between the proteins, both in amino acid sequence and relative backbone RMSD, the averaged solvent densities of water (cyan) and TFE (red) are significantly different at this region. This difference illustrates that neighboring effects on solvent density from nonidentical residues extend over many angstroms, and that a region with conserved amino acid sequence does not necessarily indicate a region with conserved solvent interactions.